A novel method for the production of stabile vaccines

ABSTRACT

The present invention relates to a method for producing stabilised vaccines, the method comprising: (a) mixing antigens with a solution comprising: (i) chitosan; (ii) at least three different amino acids and/or at least one dipeptide or tripeptide; and (iii) a sugar; and (b) drying the mixture obtained in (a).

The present invention relates to a method for producing stabilisedvaccines, the method comprising: (a) mixing antigens with a solutioncomprising: (i) chitosan; (ii) at least three different amino acidsand/or at least one dipeptide or tripeptide; and (iii) a sugar; and (b)drying the mixture obtained in (a).

In this specification, a number of documents including patentapplications and manufacturer's manuals are cited. The disclosure ofthese documents, while not considered relevant for the patentability ofthis invention, is herewith incorporated by reference in its entirety.More specifically, all referenced documents are incorporated byreference to the same extent as if each individual document wasspecifically and individually indicated to be incorporated by reference.

Today, major challenges in the field of vaccination are to developthermostable vaccines and to establish needle-free vaccinationstrategies to fight viral diseases, even in developing countries [1],which also is one of the strategic aims of the WHO Global ImmunizationVision and Strategy [2]. Furthermore, to be better prepared for pandemicoutbreaks, stockpiles of stable vaccine formulations are required [3].For example, in 2011, 16 million doses of the anti-influenza A vaccinesthat were produced in prospect for a possible H1N1 outbreak had to bedestroyed by the German government due to limited shelf live. The costsfor the unused doses were immense.

Liquid preparations face a high risk of instability during storagebecause of high molecular mobility and increased likelihood of chemicalreactions and physical instability [4]. For this reason, many liquidvaccine preparations need to be stored and transported underrefrigeration and have a short shelf life. For liquid antigenpreparations, stability is often enhanced by selection of pH-bufferingsalts and the use of amino acids for stabilisation [5, 6].

The stability of such preparations, especially their thermal stability,can be greatly increased by drying the antigens. Thus, one approach toenhance storage stability that is commonly applied is freeze-drying,thereby minimising molecular mobility and the risk of intermolecularreactions. This approach requires that the antigen is stabilised duringboth the step of freezing (cryoprotection) and the subsequent step ofdrying (lyoprotection) [7]. This can, for example, be achieved by anexchange of water with other hydrophilic molecules which may replace itas hydrogen bond forming partner. Another possibility is the formationof a sugar glass matrix, which can also be used to stabilise vaccinepreparations [8].

This general principle can also be transferred to other dryingtechniques, such as spray-freeze drying or spray-drying. Spray-drying isoften the method of choice, as it avoids the freezing step and requireslower energy costs as compared to lyophilisation. Spray-drying has alsobeen shown to be a particularly advantageous drying procedure that issuitable for biomolecules, due to the short contact time with hightemperature and its special process control [9-12]. Thus, spray-dryingbeing a process resulting in a dispersible dry powder in just one stepis often favoured to freeze drying for biomolecules [13]. The productsobtained can be designed to have good dispersion characteristics as wellas low agglomeration and adhesive tendencies to allow powder handling,packaging and efficient re-dispersion upon contact with water or buffer.This is especially true for spray drying which, accordingly, isparticularly suitable for bulk production of large amounts of vaccines[14]. However, antigens processed by spray-drying are exposed to hightemperatures and mechanical stress, which may potentially result in theloss of molecular integrity and efficacy. Therefore, antigen protectionis nonetheless required even during this one-step production approach.

Another hurdle in vaccine production is the sterilisation of vaccinepreparations without significant loss of material. Standard sterilefiltrations of biomolecules leave the risk of contamination duringaseptic fill and finish [15] and may hence lead to significant economicloss. Therefore, the need for technologies that enable terminalsterilisation of biomolecules increases [16-18]. Currently, irradiationis not considered a valid sterilisation protocol for biologics since itis associated with high energy input and increases the risk for chemicaland physical modifications entailing misfolding, formation of aggregatesand fragmentation [19-21]. Especially, aggregates may lead to modifiedimmunogenicity of therapeutically applied biologics [22]. Othersterilisation techniques such as heat sterilisation or autoclaving areinappropriate due to extensive heat stress of the product.

Overall, the development of highly stable terminally sterilised powdervaccines, including split virus vaccines such as e.g. influenza A, hasnot yet been achieved. The main bottlenecks in this development are toobtain sufficient amounts of vaccine powder, to sterilise the vaccinepowder, to avoid unappreciated antigen modifications and to avoid lossof specific immunogenicity and thus efficacy.

Accordingly, despite the fact that a lot of research has been investedinto providing suitable methods for improving the stability of vaccinesduring production, sterilisation and storage, there is still a need toprovide improved methods for producing stabilised vaccines that can bestored for prolonged periods of time and under various stressconditions.

This need is addressed by the provision of the embodiments characterisedin the claims.

Accordingly, the present invention relates to a method for producingstabilised vaccines, the method comprising: (a) mixing antigens with asolution comprising: (i) chitosan; (ii) at least three different aminoacids and/or at least one dipeptide or tripeptide; and (iii) a sugar;and (b) drying the mixture obtained in (a).

In accordance with the present invention, a method is provided forproducing vaccines that are stable, in particular during the production,sterilisation and storage process. The term “stabilised”, in accordancewith the present invention, relates to the full or partial maintenanceof the biological, immunogenic activity of the vaccine, i.e. its abilityto stimulate the immune system of a subject, such as e.g. a humansubject, to recognize it as foreign, destroy it, and subsequently enablethe immune system to protect the subject against the disease for whichthe vaccine has been developed. The vaccine is considered to be stableif it maintains at least 50% of the activity that the antigen employedfor producing the vaccine has prior to being employed in the claimedproduction method. More preferably, the vaccine maintains at least 60%,such as e.g. at least 70%, more preferably at least 80%, even morepreferably at least 90%, such as e.g. at least 95% of the activity thatthe antigen employed for producing the vaccine has prior to beingemployed in the claimed production method. More preferably, the vaccinemaintains at least 98%, such as e.g. at least 99% and most preferably100% of the activity that the antigen employed for producing the vaccinehas prior to being employed in the claimed production method. Means totest whether a vaccine is stabilised are well known to the skilledperson and include, without being limiting, testing in haemagglutinationassays (HA), radioimmunoassays, biosensor analysis, immunoprecipitationor ELISA assaying. Exemplary methods are shown in the appended examples.

In a first step of the method of producing stabilised vaccines of thepresent invention, antigens are mixed with a solution comprising therecited stabilising excipients.

Suitable antigens for vaccine preparation are well known in the art andthe considerations for choosing an antigen for vaccine productioncommonly applied in the art apply mutatis mutandis with regard tochoosing a suitable antigen for vaccine production in accordance withthe present invention. Accordingly, antigens already available in theart, as well as novel antigens, may be employed in the claimed method.

Typically, antigens are proteins and polysaccharides, but may also belipids or nucleic acids, which are, however, only antigenic whencombined with proteins and polysaccharides. Antigens are often derivedfrom parts of bacteria, viruses, and other microorganisms, such as e.g.their coats, capsules, cell walls, flagella, fimbrae, or toxins.Antigens can also be non-microbial, such as e.g. self-antigens orexogenous (non-self) antigens such as pollen, egg white, or proteinsfrom transplanted tissues/organs or on the surface of transfused bloodcells.

In accordance with the present invention, the term “antigens” includes,without being limiting, (i) antigens represented by one particularmolecular type of antigen, such as e.g. one particular protein; (ii)antigen mixtures of different molecular types of antigen, such as e.g. amixture of different proteins or a mixture of proteins withpolysaccharides; as well as (iii) antigen preparations comprisingfurther components, such as e.g. in split-virus antigens, which arepreparations wherein a virus has been disrupted by e.g. a detergent, oranother method, without further removal of other viral components.

Accordingly, antigens may for example be subunit antigens, virus likeparticles, life viruses as well as viral vectors such as e.g. MVA orAdenovirus.

In accordance with the present invention, the antigens are mixed with asolution comprising the recited recipients. This solution can be anaqueous or a non-aqueous solution. In the context of the presentinvention, the term “aqueous solution” refers on one hand to water butextends on the other hand also to buffered solutions and hydrophilicsolvents miscible with water, thus being able to form a uniform phase.Examples for aqueous solutions comprise, but are not limited to water,methanol, ethanol or higher alcohols as well as mixtures thereof.Examples for non-aqueous solvents comprise, without being limiting,dimethylsulfoxide (DMSO), ethylbenzene, and other polar solvents.

Preferably, the solution is an “aqueous solution” and, more preferably,the solvent in the solution in accordance with the method of theinvention is water.

The term “comprising” in the context of the solution according to themethod of the invention denotes that further components can be presentin the solution. Non-limiting examples of such further componentsinclude saponines, as described herein below, as well as e.g., water,buffers or solvents. Preferably, the solution does not contain anyproteins. More preferably, the solution consists of: (i) chitosan-HCl;(ii) at least three different amino acids and/or at least one dipeptideor tripeptide; (iii) a sugar and (iv) at least one saponine. Even morepreferably, the solution consists of: (i) chitosan-HCl; (ii) at leastthree different amino acids and/or at least one dipeptide or tripeptide;and (iii) a sugar. It is further preferred that the solution has a pHvalue in the range of 6 to 8, more preferably the solution has a pHvalue of 7.

Chitosan is a polysaccharide composed of randomly distributedβ-(1-4)-linked D-glucosamine (deacetylated unit) andN-acetyl-D-glucosamine (acetylated unit). Chitosan is producedcommercially by deacetylation of chitin, which is the structural elementin the exoskeleton of crustaceans and the cell walls of fungi. Theprocess causes changes in molecular weight and a degree of deacetylationof the product and degradation of nutritionally valuable proteins. Themolecular weight of chitosan is between 3800 and 20.000 Daltons. Thedegree of deacetylation (% DD) can be determined by NMR spectroscopy,and the % DD in commercial chitosans ranges from 60 to 100%. Chitosancan be obtained from commercial suppliers, such as e.g. Heppe MedicalChitosan GmbH, FMC Biopolymer, Sigma Aldrich and others.

The term chitosan, as used herein, encompasses salts and derivatives ofchitosan, such as e.g. the salts chitosan-HCl, chitosan-glutamate,chitosan-aspartate, chitosan-citrate, chitosan-acetate,carboxymethyl-chitosan and chitosan derivatives such as trimethylchitosan, zwitterionic chitosan, and glycated chitosan.

It will be appreciated that the chitosan has to be present in solubleform in the solution. The skilled person knows how to choose a suitablechitosan depending on the solution employed. For example, where thesolution is an aqueous solution, or where the solution has a pH of 7,the preferred chitosan to be employed is chitosan-HCl.

Preferred amounts of chitosan, and in particular of chitosan-HCl, to beemployed are between 0.01 and 15 mg/ml, preferably between 0.1 and 10mg/ml, more preferably between 0.5 and 5 mg/ml, even more preferablybetween 1 and 3 mg/ml and most preferably the amount is 2 mg/ml.

The term “amino acid”, in accordance with the present invention, relatesto organic molecules that have a carboxylic acid group, an amino groupand a side-chain that varies between different amino acids. Amino acidsare the essential building blocks of proteins. In accordance with thepresent invention, the term “amino acid” refers to free amino acidswhich are not bound to each other to form oligo- or polymers such asdipeptides, tripeptides, oligopeptides or proteins (also referred toherein as polypeptides).

The amino acids comprised in the solution of the present invention canbe selected from naturally occurring amino acids as well as artificialamino acids or derivatives of these naturally occurring or artificialamino acids.

Naturally occurring amino acids are e. g. the 20 proteinogenic aminoacids glycine, proline, arginine, alanine, asparagine, aspartic acid,glutamic acid, glutamine, cysteine, phenylalanine, lysine, leucine,isoleucine, histidine, methionine, serine, valine, tyrosine, threonineand tryptophan. Other naturally occurring amino acids are e. g.carnitine, creatine, creatinine, guanidinoacetic acid, ornithine,hydroxyproline, homocysteine, citrulline, hydroxylysine or beta-alanine.Artificial amino acids are amino acids that have a different side chainlength and/or side chain structure and/or have the amine group at a sitedifferent from the alpha-C-atom. Derivates of amino acids include,without being limiting, n-acetyl-tryptophan, phosphonoserine,phosphonothreonine, phosphonotyrosine, melanin, argininosuccinic acidand salts thereof and DOPA. In connection with the present invention,all the terms also include the salts of the respective amino acids.

In accordance with the present invention, three or more amino acids,which differ from each other, are comprised in the solution. Forexample, the term “at least three different amino acids” also relates toat least four different amino acids, such as at least five, at leastsix, at least seven, at least eight, at least nine, at least tendifferent amino acids or more, such as at least eleven, at least 12, atleast 13, at least 14, at least 15, at least 16, at least 17 or at least18 different amino acids. The term further encompasses exactly three,exactly four, exactly five, exactly six, exactly seven, exactly eight,exactly nine, exactly ten, exactly eleven, exactly 12, exactly 13,exactly 14, exactly 15, exactly 16, exactly 17 or exactly 18 differentamino acids. It will be readily understood by a person skilled in theart that when referring to an amino acid herein, more than one moleculeof said amino acid are intended. Thus, the recited amount of differentamino acids is intended to limit the amount of different types of aminoacids, but not the number of molecules of one type of amino acid. Forexample the term “three different amino acids”, refers to threedifferent types of amino acids, wherein the amount of each individualamino acid is not particularly limited. Preferably, the number ofdifferent amino acids does not exceed 18 different amino acids.

The term “dipeptide or tripeptide”, as used herein, relates to peptidesconsisting of two or three amino acids, respectively. Exemplarydipeptides are glycylglutamine (Gly-Gln), glycyltyrosine (Gly-Tyr),alanylglutamine (Ala-Gln) and glycylglycine (Gly-Gly). Furthernon-limiting examples of naturally occurring dipeptides are carnosine(beta-alanyl-L-histidine), N-acetyl-carnosine(N-acetyl-(beta-alanyl-L-histidine), anserine (beta-alanyl-N-methylhistidine), homoanserine (N-(4-aminobutyryl)-L-histidine), kyotorphin(L-tyrosyl-L-arginine), balenine (or ophidine) (beta-alanyl-N tau-methylhistidine), glorin (N-propionyl-γ-L-glutamyl-L-ornithine-δ-lac ethylester) and barettin (cyclo-[(6-bromo-8-en-tryptophan)-arginine]).Examples of artificial dipeptides include, without being limiting,aspartame (N-L-a-aspartyl-L-phenylalanine 1-methyl ester) andpseudoproline.

Exemplary tripeptides are glutathione (γ-glutamyl-cysteinyl-glycine) andits analogues ophthalmic acid (L-γ-glutamyl-L-α-aminobutyryl-glycine) aswell as norophthalmic acid (y-glutamyl-alanyl-glycine). Furthernon-limiting examples of tripeptides include isoleucine-proline-proline(IPP), glypromate (Gly-Pro-Glu), thyrotropin-releasing hormone (TRH,thyroliberin or protirelin: L-pyroglutamyl-L-histidinyl-L-prolinamide),melanostatin (prolyl-leucyl-glycinamide), leupeptin(N-acetyl-L-leucyl-L-leucyl-L-argininal) and eisenin (pGlu-Gln-Ala-OH).It is preferred that the at least one di- or tripeptide and morepreferred all di- or tripeptides, when used in connection with medicalapplications, do not exert any pharmacological properties.

In accordance with the present invention, the solution mayalternatively, or additionally, comprise one or more di- or tripeptides.The term “at least one dipeptide or tripeptide” also relates to at leasttwo di- or tripeptides, such as at least three, at least four, at leastfive, at least six, at least seven, at least eight or at least nine di-or tripeptides. The term further encompasses exactly one, exactly two,exactly three, exactly four, exactly five, exactly six, exactly seven,exactly eight or exactly nine di- or tripeptides. Where more than onedi- or tripeptide is comprised in the solution, a mixture of dipeptidesand tripeptides is explicitly envisaged herein. The number of di- andtripeptides can be selected independently of each other, e.g. thesolution may comprise two dipeptides and three tripeptides. It will bereadily understood by the skilled person that when referring to acertain number of di- and tripeptides herein, said number is intended tolimit the amount of different types of di- and tripeptides, but not thenumber of molecules of one type of dipeptide or tripeptide. Thus, forexample the term “four dipeptides or tripeptides”, refers to fourdifferent types of dipeptides and/or tripeptides, wherein the amount ofeach individual di- and/or tripeptide is not particularly limited.Preferably, the number of (different) di- or tripeptides does not exceednine di- or tripeptides.

Preferably, the total amount of all amino acids, dipeptides and/ortripeptides (that is the sum of all of these components in the solution)to be employed is between 0.1 and 160 mg/ml, preferably between 10 and120 mg/ml, more preferably between 40 and 100 mg/ml, even morepreferably between 60 and 90 mg/ml and most preferably the amount is 80mg/ml.

In accordance with the present invention, the solution further comprisesa sugar. Any types of sugars, i.e. the monosaccharide, disaccharide oroligosaccharide forms of carbohydrates as well as sugar alcohols, areencompassed by said term. Examples of sugars commonly used in methods ofvaccine formulations include, without being limiting, saccharose,trehalose, sucrose, glucose, lactose, sorbitol or mannitol. The solutioncomprises preferably between 0.1 mg/ml to 300 mg/ml sugar, morepreferably between 80 mg/ml to 200 mg/ml sugar, even more preferablybetween 100 mg/ml to 180 mg/ml sugar and most preferably 160 mg/mlsugar. It is explicitly envisaged that the term “a sugar” is not limitedto one type of sugar, i.e. said term also encompasses one or more typesof sugar, such as e.g. a mixture of two different types of sugar.Preferably, the term refers to only one type of sugar, such as e.g.trehalose. Where a mixture of different types of sugar is employed, theabove recited preferred amounts refer to the sum of all sugars in thesolution.

The term “dried preparation”, as used herein, refers to a preparation inwhich the liquid content has been removed or reduced. Suitable methodsfor drying an antigen preparation include, without being limiting,lyophilisation (freeze-drying), spray-drying, spray-freeze drying, airdrying or vacuum drying or supercritical drying.

The liquid content is considered to have been reduced if the liquid isreduced to less than 20% of the volume, such as for example less than10%, such as for example less than 8%, more preferably less than 7% ofthe volume, such as less than 5% or less than 1%. Even more preferably,the liquid is reduced to 0.5% or less. Most preferably, the liquid iscompletely reduced, i.e. the remaining liquid is 0% as determined bystandard methods.

In accordance with the method of the present invention, a dry vaccine isobtained. It is particularly preferred that the vaccine is a powdervaccine. In the case of spray drying, the resulting dried vaccine isobtained in the form of a powder. In those cases where the dry vaccineis not obtained as a powder, but in instead in the form of e.g. a driedcake, the skilled person is aware of how to further modify the vaccinein order to obtain a powder.

The reduced water content reduces molecular mobility within the productand hence minimises/reduces/inhibits degradation and, thus, offersadditional protection of the antigens during storage. Furthermore,surface antigens as well as proteins necessary for host cell bindingthat are present on the envelope or coat of e.g. a virus, subunitantigens, virus like particles, life viruses, viral vectors, e.g. MVA,Adenovirus etc. are protected by the inventive solution during thedrying step, as well as a potential subsequent sterilisation step, thusmaintaining the antigenicity of the antigens. Accordingly, afterreconstitution, the antigens represent vaccines.

In a further preferred embodiment of the method of the invention, themethod further comprises the step of subsequently storing the stabilisedvaccine at a temperature selected from about −90° C. to about 45° C.More preferably, the stabilised vaccine is subsequently stored at atemperature range selected from the group consisting of about −90° C. toabout −70° C., about −30° C. to about −10° C., about 1° C. to about 10°C., about 15° C. to about 25° C. and about 30° C. to about 43° C. Evenmore preferably, the stabilised vaccine is subsequently stored at atemperature range selected from the group consisting of about −85° C. toabout −75° C., about −25° C. to about −150° C., about 2° C. to about 8°C. and about 20° C. to about 40° C. Most preferably, the stabilisedviruses or bacteria are subsequently stored at a temperature selectedfrom about −80° C., about −20° C., room temperature, about 4° C. andabout 25° C.

The term “about”, as used herein, encompasses the explicitly recitedvalues as well as small deviations therefrom. In other words, atemperature of “about −90° C.” includes, but does not have to be exactlythe recited amount of −90° C. but may differ by several degrees, thusincluding for example −91° C., −92° C., −89° C. or −88° C. The skilledperson is aware that such values are relative values that do not requirea complete accuracy as long as the values approximately correspond tothe recited values. Accordingly, a deviation from the recited value offor example 15%, more preferably of 10%, and most preferably of 5% isencompassed by the term “about”.

In accordance with the present invention, a preclinical safety andefficacy study was conducted to evaluate new vaccine formulationsregarding thermal resistance, storage stability and resistance againstirradiation-mediated damage. As is shown in the appended examples, itcould surprisingly be demonstrated that vaccine formulation by drying isa feasible strategy to produce highly stable and efficacious vaccinepowders, such as e.g. influenza A vaccine powders. These vaccines wereeven shown to be suitable for terminal sterilisation (e.g.β-irradiation).

The efficacy of the novel antigen stabilizing and protecting solutions(also referred to herein as “the inventive solution”) was evaluated withregard to protection of H1N1 split virus antigen under experimentalconditions in vitro and in vivo. Original vaccine or vaccine that wasre-buffered with the inventive solution was spray-dried and terminallysterilised by irradiation with 25 kGy (e-beam) and antigen integrity wasmonitored by SDS-PAGE, dynamic light scattering, size exclusionchromatography and functional haemagglutination assays. In vitroscreening experiments revealed a number of highly stable compositionscontaining chitosan.

As discussed herein above, there are several antigen stability issues toaddress during the development of dried vaccines, in particular drypowder vaccines. For example, spray-drying is associated with hightemperature and mechanical shear stress for biomolecules, such asproteins, that may result in protein aggregation and degradation [3].Stabilizing excipients should, therefore, particularly have stabilizingefficacy against thermal stress.

Typically, before starting the development of a stable vaccinecomposition, a pre-screening by Differential Scanning Fluorimetry (DSF)is carried out to determine the stabilizing potency of stabilizingexcipients against thermal stress using a common model protein [23, 24].This fluorescence-based thermal shift assay is a reliable measure tomonitor a protein conformational stability upon thermal denaturation andan excellent to screen for conditions that stabilize a protein or anantigen. As a probe, an environmentally sensitive fluorescence dye whosequantum yield increases upon binding to increasingly exposed hydrophobicprotein regions upon unfolding is applied to monitor thermal unfoldingof a protein. The comparison of the protein melting temperature T_(m),the temperature at the equilibrium where the concentrations of foldedand unfolded protein are equal, in different stabilizing environments ofthe protein is a convenient method to evaluate the stabilising effect ofthe analysed excipient mixtures on the protein stability. Using such anapproach, it could be surprisingly shown that the addition of chitosan,here the salt chitosan-HCl, to a mixture of stabilizing excipients witha model protein resulted in a remarkable shift of the thermal profilesof the model proteins to higher temperatures, which could not beachieved with either the excipients alone or a mixture of thestabilizing excipients.

Chitosan has an antibacterial effect [34], which renders it a particularadvantageous addition to vaccine preparations during the entirepreparation process. Moreover, chitosan has previously been reported tohave an adjuvant effect [25, 27] and to improve vaccination efficacy dueto improved bio-adhesion of the vaccine on the mucosa, to increasebioavailability and to boost mucosal immune response [30-33]. Thisrenders its addition to vaccine preparations further advantageous,because less acceptable additional components such as e.g. thebactericide thiomersal or squalen as part of the adjuvant AS03 no longerneed to be included in the vaccine. Accordingly, the addition ofchitosan during the preparation process of dried vaccines not onlyavoids the use of unwanted bactericides and adjuvants, but additionallyprovides a surprising stabilising effect on the vaccine; see example 2,3, 4 and 5.

Storage data revealed high stability of protected vaccines, i.e. afterstorage of the spray dried vaccine with and without irradiation, norelevant loss of stability was monitored over three months storageperiod at 2 to 8° C. (representing normal refrigerated storageconditions) or at 25° C./60% relative humidity (representing acceleratedstability storage conditions) (example 4). Data from 3 months storage at25° C. thus provide evidence for real time storage stability of 12months at 2 to 8° C.

Additional in vivo experiments revealed that animals receiving originalvaccine exhibited the expected levels of seroconversion after 21 days(prime) and 48 days (boost) as assessed by haemagglutination inhibitionand microneutralisation assays. However, animals vaccinated withspray-dried and irradiated vaccine failed to exhibit seroconversionafter 21 days. This loss of activity could be prevented when thevaccines where protected by the inventive solution (also referred toherein as “the protected vaccines”), resulting in similar seroconversionlevels to those vaccinated with original vaccine. Boost immunisationwith protected vaccine resulted in a strong increase in seroconversionbut had only minor effects in animals treated with unprotected vaccine.

This finding is particularly surprising, as chitosan has been describedin the art as being particularly instable when exposed to radiation,such as e.g. γ-radiation [28]. As shown in the appended examples,chitosan nonetheless was found to provide a stabilising effect duringthe production and subsequent sterilisation of antigens.

Thus, it could be shown that spray-drying and terminal sterilisation ofvaccines, such as e.g. H1N1 split virus vaccine, is feasible in thepresence of the protective solution of the invention. To the inventors'best knowledge, no successful preclinical data with terminallysterilised dry-powder influenza A vaccine has been reported so far.These findings indicate the potential utility of such formulatedvaccines e.g. for needle-free vaccination routes and delivery tocountries with uncertain cold chain facilities, thereby overcoming thepresently encountered major drawbacks in vaccine manufacturing anddistribution, i.e. the need for cooled storage and the impossibility ofterminal sterilisation.

In an alternative embodiment, the present invention relates to a methodfor producing stabilised (poly)peptides, the method comprising: (a)mixing (poly)peptides with a solution comprising: (i) chitosan; (ii) atleast three different amino acids and/or at least one dipeptide ortripeptide; and (iii) a sugar; and (b) drying the mixture obtained in(a).

All definitions and preferred embodiments described herein with regardto a method for producing stabilised vaccines apply mutatis mutandis tothis alternative embodiment for producing stabilised (poly)peptides.

The term “(poly)peptide” in accordance with the present inventiondescribes a group of molecules which comprises the group of peptides,consisting of up to 30 amino acids, as well as the group ofpolypeptides, consisting of more than 30 amino acids. Also encompassedby the term “(poly)peptide” are proteins as well as fragments ofproteins. (Poly)peptides may form dimers, trimers and higher oligomers,i.e. consisting of more than one (poly)peptide molecule. (Poly)peptidemolecules forming such dimers, trimers etc. may be identical ornon-identical. The corresponding higher order structures are,consequently, termed homo- or heterodimers, homo- or heterotrimers etc.Homo- or heterodimers etc. also fall under the definition of the term“(poly)peptide”. The terms “polypeptide” and “protein” are usedinterchangeably herein. The term “(poly)peptide” also refers tonaturally modified (poly)peptides wherein the modification is effectede.g. by post-translational modifications such as e.g. glycosylation,acetylation, phosphorylation and the like. Such modifications are wellknown in the art.

In a preferred embodiment, the (poly)peptide is selected from the groupconsisting of therapeutic proteins, like antibodies, growth factors,cytokines, protein or peptide hormones, growth hormones, blood factors,therapeutic enzymes, therapeutic vaccines or fragments thereof whichretain their biological activity. These (poly)peptides can be used, forexample, for therapeutic or diagnostic purposes. Such purposes are wellknown in the art.

The term “antibody” includes polyclonal or monoclonal antibodies as wellas derivatives thereof which retain their binding specificity. The termalso includes synthetic, chimeric, single chain and humanized antibodiesor derivatives or fragments thereof, which still retain their bindingspecificity. Fragments of antibodies comprise, inter alia, Fabfragments, F(ab′)₂ or Fv fragments. Techniques for the production ofantibodies and fragments thereof are well known in the art anddescribed, e.g. in Harlow and Lane “Antibodies, A Laboratory Manual”,Cold Spring Harbor Laboratory Press, 1988 and Harlow and Lane “UsingAntibodies: A Laboratory Manual” Cold Spring Harbor Laboratory Press,1998. Further, transgenic animals may be used to express humanizedantibodies. Most preferably, the antibody is a monoclonal antibody. Forthe preparation of monoclonal antibodies, any technique that providesantibodies produced by continuous cell line cultures can be used.Examples for such techniques include the hybridoma technique (Köhler andMilstein Nature 256 (1975), 495-497), the trioma technique, the humanB-cell hybridoma technique (Kozbor, Immunology Today 4 (1983), 72) andthe EBV-hybridoma technique to produce human monoclonal antibodies (Coleet al., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc.(1985), 77-96). The antibody may be any class of antibody. It is mostpreferred that the antibody is monoclonal and of the IgG, IgM or IgYclass. IgY antibodies represent the analogs of IgG antibodies inchicken.

The term “growth factor” as used herein refers to proteins that bind toreceptors on the cell surface, with the primary result of activatingcellular proliferation and/or differentiation. Many growth factors areversatile, stimulating cellular division in numerous different celltypes, while others are specific to a particular cell-type. Preferredgrowth factors in accordance with the present invention include, withoutbeing limiting, erythropoietin, insulin-like growth factor 1 (IGF-1,originally called Somatomedin C) and insulin-like growth factor 2(IGF-2).

The term “cytokine”, in accordance with the present invention, relatesto a class of signalling proteins that are used extensively in cellularcommunication, immune function and embryogenesis. Cytokines are producedby a variety of hematopoietic and non-hematopoietic cell types and canexert autocrine, paracrine and endocrine effects as do the hormones.However, many cytokines exhibit growth factor activity. Cytokine are aunique family of growth factors. Secreted primarily from leucocytes,cytokines stimulate both the humoral and cellular immune responses aswell as the activation of phagocytic cells. Cytokines that are secretedfrom lymphocytes are termed lymphokines, whereas those secreted bymonocytes or macrophages are termed monokines. A large family ofcytokines are produced by various cells of the body. Many of thelymphokines are known as interleukines (ILs), since they are not onlysecreted by leukocytes but also able to affect the cellular responses ofleukocytes. Specifically, interleukines are growth factors targeted tocells of hematopoietic origin. Cytokines include, without beinglimiting, interleukins, interferons, granulocyte colony-stimulatingfactor (G-CSF), granulocyte-macrophage colony-stimulating factor(GM-CSF) and bone morphognetic protein-2 (BMP-2).

The term “protein or peptide hormones” refers to a class of peptidesthat are secreted into the blood stream and have endocrine functions inliving animals. Like other proteins, peptide hormones are synthesized incells from amino acids according to an mRNA template, which is itselfsynthesized from a DNA template inside the cell nucleus. Peptide hormoneprecursors (pre-prohormones) are then processed in several stages,typically in the endoplasmic reticulum, including removal of theN-terminal signal sequence and sometimes glycosylation, resulting inprohormones. The prohormones are then packaged into membrane-boundsecretory vesicles, which can be secreted from the cell by exocytosis inresponse to specific stimuli e.g. increase of calcium and cAMPconcentration in cytoplasm. These prohormones often contain superfluousamino acid residues that are needed to direct folding of the hormonemolecule into its active configuration but have no function once thehormone folds. Specific endopeptidases in the cell cleave the prohormonejust before it is released into the bloodstream, generating the maturehormone form of the molecule. Mature peptide hormones then diffusethrough the blood to all of the cells of the body, where they interactwith specific receptors on the surface of their target cells. Somepeptide/protein hormones (angiotensin II, basic fibroblast growthfactor-2, parathyroid hormone-related protein) also interact withintracellular receptors located in the cytoplasm or nucleus by anintracrine mechanism. Several important peptide hormones are secretedfrom the pituitary gland. The anterior pituitary secretes prolactin,which acts on the mammary gland, adrenocorticotrophic hormone (ACTH),which acts on the adrenal cortex to regulate the secretion ofglucocorticoids, and growth hormone, which acts on bone, muscle, and theliver. The posterior pituitary gland secretes antidiuretic hormone, alsocalled vasopressin, and oxytocin. Peptide hormones are produced by manydifferent organs and tissues, however, including the heart(atrial-natriuretic peptide (ANP) or atrial natriuretic factor (ANF))and pancreas (insulin and somatostatin), the gastrointestinal tractcholecystokinin, gastrin), and adipose tissue stores (leptin). Someneurotransmitters are secreted and released in a similar fashion topeptide hormones, and some ‘neuropeptides’ may be used asneurotransmitters in the nervous system in addition to acting ashormones when released into the blood. When a peptide hormone binds toreceptors on the surface of the cell, a second messenger appears in thecytoplasm, which triggers intracellular responses. Peptide hormonesinclude without being limited Insulin, Glucagon, Gonadotropin, humanThyroid Stimulating Hormone, angiotensin II, basic fibroblast growthfactor-2, parathyroid hormone-related protein, vasopressin, oxytocin,atrial-natriuretic peptide (ANP) or atrial natriuretic factor (ANF),somatostatin, cholecystokinin, gastrin, and adipose tissue stores(leptin).

The term “growth hormone” (GH) refers to a protein-based peptide hormoneconsisting of a 191-amino acid, single-chain polypeptide, stored andsecreted by somatotroph cells within the lateral wings of the anteriorpituitary gland. Effects of growth hormone on the tissues of the bodycan generally be described as anabolic (building up). Like most otherprotein hormones, it acts by interacting with a specific receptor on thesurface of cells. Increased height during childhood is the most widelyknown effect of GH. Height appears to be stimulated by at least twomechanisms: 1. because polypeptide hormones are not fat-soluble, theycannot penetrate sarcolemma. Thus, GH exerts some of its effects bybinding to receptors on target cells, where it activates the MAPK/ERKpathway. Through this mechanism GH directly stimulates division andmultiplication of chondrocytes of cartilage. 2. GH also stimulates,through the JAK-STAT signaling pathway, the production of insulin-likegrowth factor 1 (IGF-1, formerly known as somatomedin C), a hormonehomologous to proinsulin. The liver is a major target organ of GH forthis process and is the principal site of IGF-1 production. IGF-1 hasgrowth-stimulating effects on a wide variety of tissues. AdditionalIGF-1 is generated within target tissues, making it what appears to beboth an endocrine and an autocrine/paracrine hormone. IGF-1 also hasstimulatory effects on osteoblast and chondrocyte activity to promotebone growth. In addition to increasing height in children andadolescents, growth hormone has many other effects on the body:increases calcium retention, and strengthens and increases themineralization of bone, increases muscle mass through sarcomerehyperplasia, promotes lipolysis, increases protein synthesis, stimulatesthe growth of all internal organs excluding the brain, plays a role inhomeostasis, reduces liver uptake of glucose, promotes gluconeogenesisin the liver, contributes to the maintenance and function of pancreaticislets, stimulates the immune system.

Somatotropin refers to the growth hormone 1 produced naturally inanimals, whereas the term somatropin refers to growth hormone producedby recombinant DNA technology, and is abbreviated “HGH” in humans. Itstimulates growth, cell reproduction and regeneration.

The term “blood factors” refers to proteins that govern the functions ofthe blood coagulation cascade. The coagulation cascade of the human bodycomprises of a series of complex biochemical reactions, which areregulated by the blood factor proteins. These proteins include, forexample, the pro-coagulation factors, such as Factor VIII and Factor IX,as well as anticoagulation factors, including Protein C and AntithrombinIII.

As used herein, the term “therapeutic enzymes” refers to proteins thatcatalyse chemical reactions, thereby converting a starting molecule, thesubstrate, into a different molecule, the product. The function oftherapeutic enzymes depends directly on their molecular structure andconformation. Irreversible conformational changes and irreversibleaggregation lead to inactivation of the therapeutic enzymes. Preferredenzymes in accordance with the present invention include, without beinglimiting, therapeutic enzymes for the treatment of lysosomal storagediseases by enzyme replacement therapy, i.e. human β-glucocerebrosidase(Gaucher disease), human galactosidase A (Fabry disease), thrombolyticdrugs, i.e. sreptokinase (thrombolytic agent in treatment of ischemicstroke), urokinase, (recombinant) tissue plasminogen activator, TNKase;L-asparaginase (cytostatic drug), urate oxidase, papain.

A large number of suitable methods exist in the art to produce(poly)peptides. For example, (poly)peptides may be produced inappropriate hosts. If the host is a unicellular organism such as aprokaryote, a mammalian or insect cell, the person skilled in the artcan revert to a variety of culture conditions. Conveniently, theproduced (poly)peptide is harvested from the culture medium, lysates ofthe cultured organisms or from isolated (biological) membranes byestablished techniques. In the case of a multicellular organism, thehost may be a cell which is part of or derived from a part of theorganism, for example said host cell may be the harvestable part of aplant. A preferred method involves the recombinant production of(poly)peptides in hosts as indicated above. For example, nucleic acidsequences encoding the (poly)peptide to be folded/prevented fromunfolding according to the invention can be synthesized by PCR andinserted into an expression vector. Subsequently a suitable host may betransformed with the expression vector. Thereafter, the host is culturedto produce the desired (poly)peptide(s), which is/are isolated and,optionally, purified before use in the method of the invention.

An alternative method for producing the (poly)peptide to be employed inthe method of the invention is in vitro translation of mRNA. Suitablecell-free expression systems include rabbit reticulocyte lysate, wheatgerm extract, canine pancreatic microsomal membranes, E. coli S30extract, and coupled transcription/translation systems such as theTNT-system (Promega). These systems allow the expression of recombinant(poly)peptides upon the addition of cloning vectors, DNA fragments, orRNA sequences containing coding regions and appropriate promoterelements.

In addition to recombinant production, the (poly)peptide to be employedin the method of the invention may be produced synthetically, e.g. bydirect peptide synthesis using solid-phase techniques (cf Stewart et al.(1969) Solid Phase Peptide Synthesis; Freeman Co, San Francisco;Merrifield, J. Am. Chem. Soc. 85 (1963), 2149-2154). Synthetic peptidesynthesis may be performed using manual techniques or by automation.Automated synthesis may be achieved, for example, using the AppliedBiosystems 431A Peptide Synthesizer (Perkin Elmer, Foster City Calif.)in accordance with the instructions provided by the manufacturer.Various fragments may be chemically synthesized separately and combinedusing chemical methods to produce the full length molecule. As indicatedabove, chemical synthesis, such as the solid phase procedure describedby Houghton Proc. Natl. Acad. Sci. USA (82) (1985), 5131-5135, can beused. Furthermore, the (poly)peptide may be produced semi-synthetically,for example by a combination of recombinant and synthetic production.

(Poly)peptide isolation and purification can be achieved by any one ofseveral known techniques; for example and without limitation, ionexchange chromatography, gel filtration chromatography and affinitychromatography, high pressure liquid chromatography (HPLC), reversedphase HPLC, and preparative disc gel electrophoresis.

In a preferred embodiment of the method of the invention, the at leastthree amino acids are selected from the groups of (a) amino acids withnon polar, aliphatic R groups; (b) amino acids with polar, uncharged Rgroups; (c) amino acids with positively charged R groups; (d) aminoacids with negatively charged R groups; and (e) amino acids witharomatic R groups.

The naturally occurring amino acids, but also other than naturallyoccurring amino acids such as artificial amino acids, can be classifiedinto the above characteristic groups (Nelson D. L. & Cox M. M.,“Lehninger Biochemie” (2005), pp. 122-127), from which at least threeamino acids are selected for the solution according to the invention.

In a more preferred embodiment, the at least three amino acids areselected from different groups (a) to (e). In other words, in thispreferred embodiment, when three amino acids are comprised in thesolution, the three amino acids may be selected from at least twodifferent groups and, more preferably, from three different groups suchthat for example one is from group (a), one is from group (b) and one isfrom group (c). Further combinations such as e.g. (b)-(c)-(d),(c)-(d)-(e), (e)-(a)-(b), (b)-(d)-(e) and so forth are also explicitlyenvisaged herein. The same consideration applies when four amino acidsare comprised in the solution, in which case the amino acids have to befrom at least two different groups selected from (a) to (e), morepreferably from at least three different groups and most preferably fromfour different groups. Inter alia, when five amino acids are comprisedin the solution, the amino acids have to be from at least two differentgroups selected from (a) to (e), more preferably from at least threedifferent groups, more preferably from at least four different groupsand most preferably from five different groups. The same considerationsapply when more than five amino acids are comprised in the solution,such as e.g. six or seven amino acids, in which case these amino acidsare selected from at least two different groups selected from (a) to(e), more preferably from at least three different groups, even morepreferably from at least four different groups and most preferably fromall five different groups.

In a more preferred embodiment of the method of the invention, thesolution comprises at least one amino acid selected from each group of(a) an amino acid with non polar, aliphatic R groups; (b) an amino acidwith polar, uncharged R groups; (c) an amino acid with positivelycharged R groups; (d) an amino acid with negatively charged R groups;(e) an amino acid with aromatic R groups.

The skilled person further understands that it is not necessary that thesame number of amino acids of each group is present in the solution usedaccording to the invention. Rather, any combination of amino acids canbe chosen as long as at least one amino acids of each group is present.

In another preferred embodiment of the method of the invention, thesolution comprises at least the amino acids: (a) alanine, glutamate,lysine, threonine and tryptophane; (b) aspartate, arginine,phenylalanine, serine and valine; (c) proline, serine, asparagine,aspartate, threonine, phenylalanine; (d) tyrosine, isoleucine, leucine,threonine, valine; (e) arginine, glycine, histidin, alanine, glutamate,lysine, tryptophane, or (f) alanine, arginine, glycine, glutamate,lysine.

In accordance with this embodiment, at least the above recited aminoacids of either group (a), (b), (c), (d), (e) or (f) are present in thesolution in accordance with the invention. In other words, whereas morethan the above recited amino acids may be comprised in the inventivesolution, it is required that at least the recited amino acids arepresent. More preferably, the solution comprises exactly the recitedamino acids and no other amino acids.

In a further preferred embodiment of the method of the invention, one ormore of the amino acids are selected from the group consisting ofnatural non-proteinogenic amino acids and synthetic amino acids.

The term “non-proteinogenic amino acids”, in accordance with the presentinvention, relates to amino acids that are not naturally incorporatedinto polypeptides and proteins. Non-proteinogenic amino acids can bederived from proteinogenic amino acids, which are L-α-amino acids, bypost-translational modifications. Such non-proteinogenic amino acidsare, for example, lanthionine, 2-aminoisobutyric acid, dehydroalanine,and the neurotransmitter gamma-aminobutyric acid. Also the D-enantiomersof proteinogenic L-amino acids represent non-proteinogenic amino acids.Further non-limiting examples of non-proteinogenic amino acids includecarnitine, creatine, creatinine, guanidinoacetic acid, ornithine,hydroxyproline, homocysteine, citrulline, hydroxylysine or beta-alanine.

The term “synthetic amino acids”, as used herein, relates to amino acidsnot naturally occurring in nature. Non-limiting examples of syntheticamino acids include (2R)-amino-5-phosphonovaleric acid, D-phenyl glycineor (S)- and (R)-tert-leucine.

In another preferred embodiment of the method of the invention, the atleast one of the dipeptide(s) is selected from the group consisting ofcarnosin, glycyltryrosine, glycylglycine and glycylglutamine.

For the most injectable pharmaceutical formulations a physiologicalosmolality of approximately 300 mOsmol/kg is required. An osmolality ofapproximately 400 mOsmol/lkg is widely accepted. But the Osmolality of amixture of e.g. 10 components (amino acids) achieves a significantgreater Osmolality. In the most stabilizing applications the side chainsare the reactive part of the molecule associated with their protectiveefficacy. Hence the application of dipeptides containing the same sidechains can reduce the osmolality of the formulation. Furthermore, thereactivity of some functional groups may be reduced in the dipeptide.

In another preferred embodiment of the method of the invention, thesugar is trehalose.

Trehalose is a natural α-linked disaccharide formed by anα,α-1,1-glucoside bond between two α-glucose units. It is also known asmycose or tremalose. Trehalose is well known in the art and can beobtained commercially, for example from Sigma-Aldrich, as shown in theappended examples.

Preferred amounts of trehalose to be employed are as described hereinabove with regard to the amounts of sugar to be employed.

In another preferred embodiment of the method of the invention, thesolution further comprises at least one saponine.

Saponines are a class of chemical compounds forming secondarymetabolites which are found in natural sources, derived from naturalsources or can be chemically synthesised. Saponines are found inparticular abundance in various plant species. Saponines are amphipathicglycosides grouped phenomenologically by the soap-like foaming theyproduce when shaken in aqueous solutions, and structurally by theircomposition of one or more hydrophilic glycoside moieties combined witha lipophilic steroidal or triterpenoid aglycone. Their structuraldiversity is reflected in their physicochemical and biologicalproperties. Non-limiting examples of saponines are glycyrrhizic acid,glycyrrhetinic acid, glucuronic acid, escin, hederacoside and digitonin.

Preferably, the saponine is glycyrrhizic acid or a derivative thereof.Glycyrrhizic acid is also known as glycyrrhicic acid, glycyrrhizin orglycyrrhizinic acid. Glycyrrhizic acid is water-soluble and exists as ananion that can be a potential ligand to form electrostaticallyassociated complexes with cationic molecules of active ingredients.Without wishing to be bound by theory, the present inventors hypothesisethat the anionic glycyrrhizic acid forms complexes with amino acidspresent in the solution of the present invention (i.e. arginine, orlysine) through electrostatic interactions, hydrogen bonds or both. Thiscomplex-formation is thought to enhance the ability of the solution ofthe present invention to stabilise the vaccine during drying andstorage. Moreover, the ability of glycyrrhizic acid to form complexeswith cationic molecules of active ingredients can lead to stabilisinginteractions with exposed cationic side chains on the protein surfaceduring the storage process.

Derivatives of glycyrrhizic acid are well-known in the art and includethose produced by transformation of glycyrrhizic acid on carboxyl andhydroxyl groups, by conjugation of amino acid residues into thecarbohydrate part or the introduction of2-acetamido-β-D-glucopyranosylamine into the glycoside chain ofglycyrrhizic acid. Other derivatives are amides of glycyrrhizic acid,conjugates of glycyrrhizic acid with two amino acid residues and a free30-COOH function and conjugates of at least one residue of amino acidalkyl esters in the carbohydrate part of the glycyrrhizic acid molecule.Examples of specific derivatives can be found e. g. in Kondratenko etal. (Russian Journal of Bioorganic Chemistry, Vol 30(2), (2004), pp.148-153).

Preferred amounts of glycyrrhizic acid (or derivatives thereof) to beemployed are between 0.01 and 15 mg/ml, preferably between 0.1 and 10mg/ml, more preferably between 0.5 and 5 mg/ml, even more preferablybetween 1 and 3 mg/ml and most preferably the amount is 2 mg/ml.

As is known in the art, saponines, in particular glycyrrhizic acid, hasbeen found to be advantageously present in stabilising formulations, asit enhances the stabilising effect of other excipients.

In a further preferred embodiment of the method of the invention, theantigens are selected from the group consisting of influenza subunitantigen, haemagglutinin, neuraminidase, cholera toxin B subunit,hepatitis B surface antigen, toxoids, HIV envelope protein, anthraxrecombinant protective antigen, other pathogen surface proteins andvirus envelope components

In a more preferred embodiment of the method of the invention, theantigens are split virus antigens.

Split-virus antigens are well known in the art and are commonly employedas a basis for vaccines (WHO, Unicef, WorldBank. State of the world'svaccines and immunization. 3rd ed. Geneva: World Health Organisation,2009.). Split-virus antigens are obtained by inactivating and disruptinga virus by e.g. a detergent or by other techniques, without furtherremoval of other viral components. Split-virus vaccines are oftenemployed for vaccination against, amongst other, influenza, hepatitis A,Japanese encephalitis, poliomyelitis or rabies. It is even morepreferred that the split virus antigens are influenza virus antigens.Influenza virus is part of the family of Orthomyxoviridae and belongs tovirus group V ((−)ssRNA). The three genera of influenza virusknown—influenza virus A, B and C—are identified by antigenic differencesin their nucleoprotein and matrix protein. Influenzavirus A infectshumans, other mammals, and birds, and causes flu pandemics;influenzavirus B infects humans and seals and influenzavirus C infectshumans and pigs.

The main antigenic structures of influenza split vaccines are theintegral membrane glycoproteins haemagglutinin (HA) and neuraminidase(NA) [3, 26]. Together with other proteins, including matrix proteins(MP1; MP2) and nucleoprotein (NP) and several minor components from themembrane lipid matrix, they form the constituents of the split vaccine.

The preparation of stable split-virus influenza vaccines is typicallydifficult, as the 3-dimensional structure of haemagglutinin containshighly hydrophobic regions that make it susceptible to form solubleaggregates and protein complexes with other influenza constituents [26].In addition, haemagglutinin is susceptible to freezing stresses,particularly sensitive to pH drops and to changes in the concentrationof solutes during freezing, leading to irreversible conformationalchanges and denaturation, while elevated temperatures can causeinactivation of the virus antigen [3]. In addition, the problem ofterminal sterilisation has not been resolved successfully so far.

In the past decades, several papers have been published in which driedinfluenza vaccines were used. However, the development of dry-stateinfluenza vaccines is still in a very early stage. Incorporation ofinfluenza vaccines in amorphous glassy carbohydrate matrices canstabilize the various antigens against different kinds of stressesassociated with different drying methods. However, for each dryingmethod and for each influenza antigen the stability of the vaccine wasdependent on the type of the chosen carbohydrate glassy matrix. Eachvaccine type may possess its own intrinsic sensitivity to differentstresses associated with the different drying methods. Many aspects ofstabilization of influenza vaccines, in particular the comparison of thedifferent drying methods for the production of stabilized influenzavaccines, had to be further investigated. As a result, the incorporationof a vaccine compound in carbohydrate glasses needs be optimized by bothformulation and drying process considerations. Additional data on longterm stability of dry influenza vaccine formulations and pre- andclinical studies are very limited. In the dry state, the long-termstability of the influenza vaccines is still very limited, especially atelevated temperatures. It was shown that the storage stability of driedinfluenza vaccines was dependent on the type of carbohydrate, the typeof buffer and storage conditions. Terminal sterilization is up to datenot considered to be a part of the production process of influenzavaccine antigens [3].

In accordance with the present invention, these problems couldsuccessfully be addressed by the method of the invention, therebypreparing a highly stable split-virus influenza vaccine powder, as shownin the appended examples. As is evident from those data, incorporationin a sugar matrix alone was not sufficient to stabilise the vaccineduring spray drying or freeze drying followed by terminal sterilisation.Further, thermal stabilisation could not be achieved with the liquidpreparation nor with a dried preparation without stabilisation using thesolution of the invention.

More preferably, the influenza virus is influenza A virus and mostpreferably, the influenza virus is influenza A H1N1 virus. Even morepreferably, the influenza A H1N1 virus is the inactivated, split virionA/California/7/2009 (H1N1)v like strain (x-179a).

In another preferred embodiment of the method of the invention, the w/wratio between the excipients of the solution and the antigen is betweenabout 1:1 and about 30.000:1.

In accordance with this embodiment, the excipients of the solution arethe non-aqueous components of the solution that are not the antigen tobe stabilised.

More preferably, the w/w ratio between the components of the solutionand the antigen is between about 1:1 and about 25.000:1, such as forexample between about 5:1 and about 20.000:1. Most preferably, the w/wratio is about 16.267:1. It will be understood that any value fallingbetween these ratios is explicitly also envisaged herein. Furthermore,the term about, as used herein, encompasses the explicitly recitedratios as well as deviations therefrom of ±10%.

In a further preferred embodiment of the method of the invention, thestep of drying the mixture is achieved by a method selected from thegroup consisting of spray drying, lyophilisation, spray-freeze drying,supercritical drying and air or vacuum drying.

Spray-drying is well known in the art and is a method to convert asolution, suspension or emulsion into a solid powder in one singleprocess step. Generally, a concentrate of the liquid product is pumpedto the atomising device, where it is broken into small droplets. Thesedroplets meet a stream of hot air and they lose their moisture veryrapidly while still dispersed in the drying air. The dry powder isseparated from the moist air in cyclones by centrifugal action, thedense powder particles are forced toward the cyclone walls while thelighter, moist air is directed away through the exhaust pipes.

Lyophilisation, also referred to as freeze-drying, is also well known inthe art and includes the steps of freezing the sample and subsequentlyreducing the surrounding pressure while adding sufficient heat to allowthe frozen water in the material to sublime directly from the solidphase to the gas phase followed by a secondary drying phase. Preferably,the lyophilised preparation is then sealed to prevent the re-absorptionof moisture.

Spray-freeze-drying is also well known in the art and is a method thatcombines processing steps common to freeze-drying and spray-drying. Thesample provide is nebulised into a cryogenic medium (such as e.g. liquidnitrogen), which generates a dispersion of shock-frozen droplets. Thisdispersion is then dried in a lyophiliser.

Supercritical drying is another technique well known in the art. Thismethod relies on high-temperature and high-pressure above the criticaltemperature (T_(c)) and critical pressure (p_(c)) to change a liquidinto a gas wherein no phase boundaries are crossed but the liquid to gastransition instead passes through the supercritical region, where thedistinction between gas and liquid ceases to apply. The densities of theliquid phase and vapor phase become equal at the critical point ofdrying.

Air drying refers to drying the sample by exposing it to the surroundingair, optionally combined with moderate heating of the sample,ventilation of the air or evacuation of the drying chamber (vacuumdrying).

In a further preferred embodiment of the method of the invention, thedried vaccine obtained in step (b) is subsequently sterilised.Preferably, the vaccine is packed in single or multiple dose containersprior to said terminal sterilisation step.

The term “sterilising” refers to a process wherein all live organismsare killed. Methods of sterilisation are well known in the art andinclude, for example, sterilisation by beta irradiation, by gammairradiation thermal sterilisation, gas sterilisation, sterilisation byethylene oxide (EO) as well as plasma sterilisation. Preferably, thesterilisation is effected by gamma- or beta-irradiation.

Preferably, the thus obtained sterile vaccine powder is subsequentlystored under sealed conditions, such as e.g. in a sealed container orvial, until its use.

As shown in the appended examples, the antigenicity of the antigenspresent in a vaccine powder produced in accordance with the method ofthis invention is maintained after sterilisation. Thus, the method ofthe present invention provides a vaccine preparation method that enablesthe preparation of stable and safe antigens, wherein the antigens arestabilised in the solution according to the invention, therebymaintaining their naturally occurring three-dimensional appearance.

In a further preferred embodiment of the method of the invention, thevaccine is for intramuscular, subcutaneous, intradermal, transdermal,oral, peroral, nasal, and/or inhalative application.

The present invention further relates to vaccine comprising (an)antigen(s), chitosan, at least three different amino acids and/or atleast one dipeptide or tripeptide, and a sugar. This vaccine has animproved stabilisation during storage and sterilisation. Preferably, thestabilised vaccine of the invention is a vaccine obtained or obtainableby the method of producing a stabilised vaccine of the presentinvention. All of the definitions and preferred embodiments with regardto the method of producing a stabilised vaccine of the present inventionapply mutatis mutandis also to the stabilised vaccine of the presentinvention.

The present invention further relates to a (poly)peptide composition,comprising (a) (poly)peptide(s), chitosan, at least three differentamino acids and/or at least one dipeptide or tripeptide, and a sugar.This (poly)peptide composition has an improved stabilisation duringstorage and sterilisation. Preferably, the stabilised (poly)peptidecomposition of the invention is a (poly)peptide composition obtained orobtainable by the method of producing a stabilised (poly)peptide of thepresent invention. All of the definitions and preferred embodiments withregard to the method of producing a stabilised (poly)peptide of thepresent invention apply mutatis mutandis also to the stabilised(poly)peptide composition of the present invention.

As regards the embodiments characterised in this specification, inparticular in the claims, it is intended that each embodiment mentionedin a dependent claim is combined with each embodiment of each claim(independent or dependent) said dependent claim depends from. Forexample, in case of an independent claim 1 reciting 3 alternatives A, Band C, a dependent claim 2 reciting 3 alternatives D, E and F and aclaim 3 depending from claims 1 and 2 and reciting 3 alternatives G, Hand I, it is to be understood that the specification unambiguouslydiscloses embodiments corresponding to combinations A, D, G; A, D, H; A,D, I; A, E, G; A, E, H; A, E, I; A, F, G; A, F, H; A, F, I; B, D, G; B,D, H; B, D, I; B, E, G; B, E, H; B, E, I; B, F, G; B, F, H; B, F, I; C,D, G; C, D, H; C, D, I; C, E, G; C, E, H; C, E, I; C, F, G; C, F, H; C,F, I, unless specifically mentioned otherwise.

Similarly, and also in those cases where independent and/or dependentclaims do not recite alternatives, it is understood that if dependentclaims refer back to a plurality of preceding claims, any combination ofsubject-matter covered thereby is considered to be explicitly disclosed.For example, in case of an independent claim 1, a dependent claim 2referring back to claim 1, and a dependent claim 3 referring back toboth claims 2 and 1, it follows that the combination of thesubject-matter of claims 3 and 1 is clearly and unambiguously disclosedas is the combination of the subject-matter of claims 3, 2 and 1. Incase a further dependent claim 4 is present which refers to any one ofclaims 1 to 3, it follows that the combination of the subject-matter ofclaims 4 and 1, of claims 4, 2 and 1, of claims 4, 3 and 1, as well asof claims 4, 3, 2 and 1 is clearly and unambiguously disclosed.

The above considerations apply mutatis mutandis to all appended claims.To give a non-limiting example, the combination of claims 12, 7 and anyone of claims 4(a) to 4(f) is clearly and unambiguously envisaged inview of the claim structure. The same applies for example to thecombination of claims 12, 8, 7 and any one of claims 4(a) to 4(f), etc.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. In case of conflict, the patentspecification, including definitions, will prevail.

The figures show:

FIG. 1: Differential Scanning Fluorimetry

Normalized thermal denaturation curves of the model protein incombination with different stabilizing excipients alone or excipientmixtures compared to the corresponding thermal denaturation profile ofthe model protein in PBS buffer. Thermal denaturation curves of themodel protein in different concentrations of trehalose (A), chitosan(B), SPS (C), SPS+trehalose (D), SPS+chitosan (E) andSPS+trehalose+chitosan (F), Comparison of the normalized thermaldenaturation curves of the analysed model protein for all excipients andexcipient mixture in a selected concentration range (G).

FIG. 2: SDS-PAGE monitoring of the selected variants over time.

Non-reducing SDS-PAGE (A) and reducing SDS-PAGE (B) of the differentformulations of the influenza vaccine before and after spray-drying andsubsequent irradiation at 25 kGy (e-beam) at the time point t=0. Thesamples are loaded for each treatment condition in the following order:Mark12 (lane 1); liquid SPS T final prior to SD (lane 2); SD SPSTrehalose final (lane 3); SD SPS T final 25 kGy (lane 4); originalPandemrix (positive control; lane 5); liquid M prior to SD (lane 6); SDM 25 kGy (lane 7); Mark12 (lane 8). Non-reducing SDS-PAGE (C and E) andreducing SDS-PAGE (D and F) of the different formulations of theinfluenza vaccine before and after spray-drying and subsequentirradiation at 25 kGy (e-beam) at the time point t=1 month (C, D) andt=3 months (E, F). The samples are loaded for each treatment conditionin the following order: Mark12 (lane 1); SD SPS T final 2-8° C. (lane2); SD SPS T final 25° C. 60% rH (lane 3); SD SPS T final 25 kGy 2-8° C.(lane 4); SD SPS T final 25 kGy 25° C. 60% rH (lane 5); SD M 25 kGy 2-8°C. (lane 6); SD M 25 kGy 25° C. 60% rH (lane 7); originalPandemrix+chitosan-HCl (lane 8); original Pandemrix (positive control;lane 9); Mark12 (lane 10).

FIG. 3: Immune responses in macaques to Pandemrix vaccine. A,Haemagglutination inhibition assay (HAI) titre; B, Microneutralisationassay (MN) titre. In each chart, bars represent the geometric mean titrefor each group. Group 1: negative control group (PBS); Group 2: originalPandemrix; Group 3: SD M 25 kGy; Group 4: SD SPS T final; Group 5: SDSPS T final 25 kGy. *, significant difference from group 2, 4 and 5means, p<0.01. Means for groups 2, 4 and 5 are not significantlydifferent from one another at any time-point.

FIG. 4: Dynamic light scattering. Particle size distribution by dynamiclight scattering of A) original Pandemrix; SD M; SD M 25 kGy; SD SPS Tfinal; SD SPS T final 25 kGy B) SD M 25 kGy, prepared freshly and after1 and 3 month storage at 2-8° C. and 25° C., 60% rH C) SD SPS T final;prepared freshly and after 1 and 3 month storage at 2-8° C. and 25° C.,60% rH D) SD SPS T final 25 kGy, prepared freshly and after 1 and 3month storage at 2-8° C. and 25° C., 60% rH

FIG. 5: SEC-Analyses. SEC chromatograms of A) original Pandemrix; SD M;SD M 25 kGy; SD SPS T final; SD SPS T final 25 kGy B) SD M 25 kGy,prepared freshly and after 1 and 3 month storage at 2-8° C. and 25° C.,60% rH C) SD SPS T final; prepared freshly and after 1 and 3 monthstorage at 2-8° C. and 25° C., 60% rH D) SD SPS T final 25 kGy, preparedfreshly and after 1 and 3 month storage at 2-8° C. and 25° C., 60% rH.

FIG. 6: Water content in % of different formulations after spray dryingas determined by Karl-Fischer titration. Results are shown as mean±SDvalues (n=3).

FIG. 7: LDH enzyme activities normalized in % of a standard activity in4 SPS variants upon liquid storage at a temperature of 50° C. beforestorage t=0 days and after 2 days, 4 days, 7 days and 14 days incomparison to the storage of LDH in 100 mM sodium phosphate buffer. Themeasured LDH activity was normalized to a standard curve.

FIG. 8: Comparison of the specific LDH enzyme activity before (t=0 days)and after liquid storage at the indicated time points (t=14; 16; 18 and21 days) formulated with either trehalose and chitosan (left side of thefigure) or with a mixture of 7 amino acids, trehalose and chitosan(right side of the figure).

FIG. 9: LDH enzyme activities normalized in % of a standard activitybefore (t=0) and after liquid storage of LDH formulated with the maincomponents employed in WO 2009/014774 (Pluronic F127 and rHSA) ascompared to a combination of 7 amino acids, trehalose and chitosan withthese components at 50° C. measured at the indicated time points (t=2; 4and 7 days).

The examples illustrate the invention.

EXAMPLE 1: MATERIALS AND METHODS Stabilizing and Protecting Solution(SPS)

The proprietary aqueous Stabilizing and Protecting Solution (SPS;LEUKOCARE, Munich, Germany) is composed of different small molecules(here mostly amino acids) and glycosidic excipients (here,glycyrrhizinic acid) usually provided as a stock concentration of 80mg/mL (pH 7). All components are used in pharmaceutical quality; theyare non-toxic and routinely used in parenteral solutions.

Influenza Strain

Pandemrix (Glaxo Smith Kline) was used as the antigen vaccine. Pandemrixis provided in two vials, one of which contains an influenza (splitvirion, inactivated) A/California/7/2009 (H1N1)v like strain (x-179a)that comprises the main antigen hemagglutinin. The second vial comprisesthe adjuvant, AS03 (squalene (10.69 mg per dose), DL-α-tocopherol (11.86mg) and polysorbate 80 (4.86 mg)). 15 μg/ml of the antigen dispersion(are usually mixed 1:1 with adjuvant prior to injection (3.74 μg HA perdose 500 μl). For the present study, the split viral antigens of thefirst vial were re-formulated as described, dried and sterilised. Priorto injection, said re-formulated antigen preparations were resuspendedand mixed 1:1 with adjuvant, as described above.

Differential Scanning Fluorimetry

SYPRO orange (5000× stock solution; Life Technologies (Carlsbad, Calif.)was diluted 1:1000 in different concentrations of the stabilizingexcipient mixtures or in PBS buffer to a 5× final concentration. In thenext step the model protein was diluted to 300 μg/ml and 600 μg/ml,respectively with the 5×SYPRO orange solutions of these differentconcentrations of the stabilizing excipients or with buffer andsubsequently distributed as 50 μl aliquots into the wells of a 96-wellPCR plate (4titude, Berlin). The PCR plates were sealed with a PCR film(4titude, Berlin) and 1 min centrifuged at 500 g at room temperature toavoid creating air bubbles and to collect the solution at the bottom ofthe wells. The plates were subsequently heated on a q-PCR Light-Cycler480 II (Roche) from 20 to 95° C., with a ramping rate of 3° C. min⁻¹.The set up of the filter configuration was the optimal excitationwavelength of 498 nm and emission wavelength of 610 nm for SYPRO orange.The midpoint of thermal denaturation T_(m) was calculated by fitting thedata to the Boltzmann equation using GraphPad Prism 6. The differencesin T_(m) between the control samples of the model protein in PBS and thestabilizing excipients containing formulations were calculated asthermal shift.

Formulation Variants

For in vitro testing, a range of formulation variants were spray driedof which a small number was selected for the in vivo study. A summary ofthe formulation variants and names used throughout the presentapplication are given in Table 3. Original Pandemrix was either used assupplied and mixed with the matrix component in the respective amount orin case of the SPS containing formulations it was dialysed overnightagainst SPS in the respective concentrations at pH 7, usingSlide-A-Lyzer® dialysis cassettes (cut-off 3.5 kDa; volume 3-12 mL)(Thermo Scientific, Schwerte-Geisecke, Germany). The additionalcomponents mannitol, trehalose and glycyrrhizinic acid (Sigma-Aldrich,Munich, Germany), respectively, were added afterwards. For in vivobatches, chitosan-HCl was also added (Heppe Medical Chitosan GmbH, Hallea. d. Saale, Germany).

Spray-Drying

A Büchi B-290 laboratory spray-dryer (Büchi, Flawil, Switzerland) wasused for spray-drying of the vaccine preparations (see table 4 forparameters). The dried product was collected in the product vessel usinga high performance cyclone, sealed in glass vials and was stored at 2 to8° C. until analysis. For the batches of the in vivo study, allcomponents of the spray-dryer were disinfected with 70% (V/V)isopropanol prior to use. Dried products were filled in individual dosesin pre-sterilised glass vials (Type I, 2R, PRI-PAC e.K. Eschweiler,Germany) under aseptic conditions.

Haemagglutination Assay

Dried samples were reconstituted in water (15 μg HA/mL). To determine HAtitres, 50 μL of the diluted split vaccine formulations were two-foldserially diluted in 50 μL PBS mixed with an equal volume of fresh 0.5%(W/V) chicken red blood cell suspension (Harlan Laboratories, Belton,Leicestershire, UK) in a U-bottom 96 well microtitre plate. After onehour incubation at room temperature, the plates were scored foragglutination. The titres are given as inverse of the highest dilutioncausing the agglutination of red blood cells.

Electrophoresis

Spray dried products were reconstituted in water (15 μg HA/mL). Fordenaturing conditions, the samples were prepared by mixing 12 μL of thevaccine formulation with 12 μL of the NuPAGE LDS-sample buffer 2×concentrate (Invitrogen, Darmstadt, Germany). Separation was performedat a constant voltage of 200 V and running time was ˜90 min. Gels werestained with a silver staining kit (SilverXpress silver staining kit,Invitrogen). A molecular weight standard (Novex Mark 12™ UnstainedStandard, Invitrogen) was analysed on each gel.

Irradiation Protocol

Sterilisation of the spray dried and freeze dried vaccine samples wasperformed in sealed glass vials by BGS, Saal a. d. Donau, Germany, usingβ-irradiation at 25 or 40 kGy. Samples for the in vivo study and longterm storage were β-irradiated with 25 kGy.

Long Term Storage

For the formulations chosen for the in vivo study, extensivecharacterisation directly after production and over storage wasperformed. All formulations were packed in glass vials, were sealed andstored at 2 to 8° C., or at 25° C. (+/−2° C.) at 60% (+/−5%) relativehumidity (rH), respectively. After one month and after three months,samples were analysed. Characterisation included haemagglutinationassay, DLS measurements, water content, SEC profiling andelectrophoresis.

Size Exclusion Chromatography (SEC)

Size exclusion chromatography (SEC) was performed by using anMerck-Hitachi D 7000 system (Merck-Hitachi, Darmstadt, Germany) with UVdetector at 214 nm and a 13 μm TSKgel GMPWXL SEC column (7.8×300 mm)(TOSOH Bioscience GmbH, Stuttgart, Germany). Samples were run in amobile phase of PBS pH 7.4 (flow rate 0.7 mL/min). A sample amountequivalent to 15 μg HA was used for each analysis. All measurements wereperformed in duplicates.

Dynamic Light Scattering (DLS)

Dynamic Light Scattering was performed to analyse for protein aggregatesusing a Malvern Zetasizer Nano-ZS (Malvern Instruments, Worcestershire,UK). A sample amount equivalent to 15 μg of protein (HA) was redispersedin double distilled water and was measured. All results are mean ofthree sets of 30 individual scans.

Water Content

50 mg of the spray dried product was dissolved in 1 mL ofdimethylsulfoxide (DMSO) and added to the titration flask of the KarlFischer titrator (V20, Mettler Toledo AG, Schwerzenbach, Switzerland).Titration was then carried out using Karl Fischer reagent of previouslydetermined titre (mg H₂O/mL). Water content was determined in triplicatefor each sample and was subtracted for solvent. Results are shown asmean±SD values.

Animal Study

The study was approved by the Ethical Review Process of Public HealthEngland, Porton, Salisbury, UK and the Home Office via Project LicensePPL30/2993. The study was conducted in accordance with the PHE PortonDown Quality Management System that is compliant with BS ENISO9001-2000.

Animals (Macaca fascicularis) consisted of mature adults with equaldistribution of male and female animals in each group. Twenty-six matureadult animals of either sex (age range 4 to 6 years and weight range 3.6to 5.9 kg at the start of the study) were obtained from a Home Officeaccredited breeding colony within the United Kingdom. All animals weremaintained within a conventional colony tested to be free of Herpesvirussimiae (B-virus), Mycobacterium tuberculosis (TB), Simian T-cellLymphotropic virus (STLV) and Simian immunodeficiency virus (SIV) andwere selected from a cohort of animals screened for the absence ofinfluenza antibodies.

The animals were housed in their existing social groups in pens designedin accordance with the requirements of the United Kingdom Home OfficeCode of Practice for the Housing and Care of Animals Used on ScientificProcedures (1989). Each animal was individually identified by apermanent tattoo using a unique number. Tap water and Expanded PrimateMaintenance diet (PME, Special Diet Services, UK) were available adlibitum with enrichment treats, vegetables and fruit provided on aregular basis.

Animal Groups: Group 1: Negative control (PBS) 2 animals Group 2:Original Pandemrix 6 animals Group 3: SD M 25 kGy 6 animals Group 4: SDSPS T final 6 animals Group 5: SD SPS T final 25 kGy 6 animals

Original Pandemrix consisted of the human vaccine formulation, includingthe AS03 adjuvant provided as a separate flask. The spray dried productswere supplied in single dose vials and were reconstituted with sterilewater and AS03 adjuvant immediately prior to vaccination. Each animalwas given 0.5 ml vaccine preparation containing 3.75 μg HA antigen(human adult dose). All vaccinations were given by intramuscularinjection. Control sera were taken eight days prior to vaccination, andsera were taken at 21, 34 and 48 days post-vaccination. A boosterimmunisation was given 28 days post-vaccination, with an equal dose ofthe appropriately-treated vaccine. At every sampling or vaccinationoccasion each animal was weighed, had a rectal temperature taken,superficial lymph nodes (inguinal and axillary) palpated, the site ofvaccination examined and a check was made of general physical condition.In addition, haemoglobin levels were checked at each blood samplingoccasion.

Haemagglutination Inhibition Assay (HAI)

Sera were treated with receptor-destroying enzyme (RDE, Denka SeikenCo., Japan), followed by heat-inactivation. Treated sera were thensubjected to 2-fold serial dilutions in 96-well U-bottom plates,followed by the addition of 4 HA units of virus (influenzaA/California/07/09). After incubating at room temperature, a 0.5% w/vsuspension of chicken red blood cells was added as described above forthe HA assay. The end-point was defined as the highest serum dilutionshowing complete HA inactivating activity.

Microneutralisation Assay (MN)

RDE-treated sera were subjected to 2-fold serial dilutions in 96-wellcell culture plates. The diluted sera were mixed with an equal volume ofmedium containing 100 TCID50 influenza A/California/07/09 virus. Afterincubation, 100 μl of Madin-Darby canine kidney (MDCK) cells were addedto each well. The plates were incubated for 18 to 20 hours. Cellmonolayers were washed with PBS and fixed. Presence of viral antigen wasdetected with a primary antibody to the influenza A NP protein followedby a secondary peroxidase conjugate. After staining, absorbance was readat 492 nm. The reciprocal serum dilution corresponding to the lowestdilution to be scored negative for neutralising activity is the 50%neutralisation antibody titre.

Data Analysis.

Data from animal study are depicted as geometric mean values of n=6animals per group. Minitab 15 software was used to conduct nonparametricanalyses using Mann-Whitney Rank Sum Test. Intergroup differences wereconsidered significant at p<0.01.

EXAMPLE 2: DIFFERENTIAL SCANNING FLUORIMETRY (DSF)—THERMAL SHIFT ASSAY

Thermal profiles of a model protein were monitored in the presence of anenvironmentally sensitive fluorescent dye which is highly fluorescent innon-polar environment, such as the hydrophobic sites of unfoldedproteins, compared to the aqueous solution where the fluorescence isquenched. The temperature at which a protein unfolds is measured by anincrease in the fluorescence of the applied dye with affinity tohydrophobic parts of the protein, which are exposed as the proteinunfolds. The plot of the fluorescence intensity as a function oftemperature generated a sigmoidal curve that can be described by a twostate transition. The inflection point of these transition curves(T_(m)) was calculated by fitting the curves using a Boltzmann equation.

A T_(m) 70.2° C. and 70.7° C. dependent of the protein concentration wasobtained for the model protein in PBS Buffer. Addition of trehalosealone to the protein solution resulted in a thermal shift between 2 to3° C. with a slight decrease in the higher concentration range oftrehalose (FIG. 1A, G and Table 1). In contrast, already the addition ofchitosan alone led to a more remarkable stabilizing effect on thethermal denaturation profile of the model protein. The thermal shift ofthe T_(m) with chitosan was between 3 to 5° C. with an increase withincreasing chitosan concentrations (FIG. 1B, G and table 1). The thermalshift of the model protein between 2.5 and 3.2° C. with an excipientmixture based on the SPS platform technology was approximatelycomparable with the stabilizing effect of trehalose in this assay (FIG.1C, G and table 1). After addition of chitosan to this excipient mixturebased on the SPS platform technology, a pronouncedconcentration-dependent increase of the thermal shift was found. Greaterthan 3° C. in the small concentration range to approximately 9° C. inthe higher concentration range was determined for this excipient mixture(FIG. 1E, G and table 1). In the case of the addition of trehalose tothe SPS based excipient mixture a concentration dependent increase ofthe thermal shift from 2.5° C. to 6.5° C. was found (FIG. 1D, G andtable 1). Further addition of chitosan to the mixture of SPS andtrehalose led to additional enhanced thermal shifts in a concentrationdependent manner. The T_(m) of the model protein increases withincreasing concentrations of the mixture from 2.7 to nearly 11° C.,suggesting a synergistic stabilizing effect of chitosan in combinationwith the different excipient mixtures (FIG. 1F, G and table 1). For thefurther study considering the stability of the H1N1 split vaccine, themixture of chitosan, SPS and trehalose with the highest concentrationand the corresponding highest thermal shift of 11° C. was applied.

EXAMPLE 3: STRUCTURAL ANALYSIS

To correlate the functional activity of the SPS-formulated vaccines withthe retention of structural integrity, SDS-PAGE migration patterns ofthese samples were compared with the positive control (FIG. 2).Non-reducing and reducing SDS-PAGE analysis of original Pandemrixindicated typical migration patterns for highly purified split vaccines.Non-reducing SDS-PAGE showed six separated bands in the case of theoriginal Pandemrix (FIG. 2A).

Because of the highly hydrophobic nature of the integral membraneprotein haemagglutinin and the resulting high susceptibility to formsoluble aggregates in solution and protein complexes with the otherprotein components, an assignment of the single bands to singlecomponents is very difficult. The lack of the protein bands migrating atmolecular weights of 200 kDa and between 200 and 116.5 kDa underreducing conditions indicated that the oligomeric forms observed undernon-reducing conditions were disulphide linked oligomers of HA0particularly dimers and trimers (FIGS. 2A and B). The bands atapproximately 65 kDa in the non-reducing SDS-PAGE may correspond to thelikewise disulphide linked HA0 monomers, not visualised by reducingSDS-PAGE. The band at approximately 55.4 kDa could correspond to smalleramounts of HA1 in complex with the nucleoprotein. A small band between31 and 21.5 kDa may correspond to the protein complex of HA2 and thematrix protein M1. The small amount of degradation of thedisulphide-linked HA0 and the dimer and trimer, respectively could be aconsequence of the sample preparation for the non-reducing gel—heatingof the samples for 10 min at 90° C. leading to maximum binding of SDS tothe protein (FIG. 2A). Upon the loss of the bands corresponding to thedisulphide-linked HA0 monomer and the haemagglutinin dimer and trimertwo prominent bands in the reducing SDS-PAGE may correspond to the HA1and HA2 subunits of haemagglutinin in complex with nucleoprotein andmatrix protein, respectively (FIG. 2B).

It is known from literature that nucleoproteins and matrix proteins,being present in the split vaccine formulation, can interfere with thedetection of haemagglutinin [26, 31]. In the case of the liquid SD Mbefore spray-drying the same migration pattern as the positive controlin the non-reducing as well as in the reducing SDS-PAGE was found (FIGS.2A and B). In contrast, SD M showed a considerable loss of bandintensity corresponding to the main antigenic components of the splitvaccine (data not shown), matching the pronounced loss of functionalintegrity in the HA-assay. Subsequent β-irradiation of this formulationat 25 kGy led to almost complete loss of the typical migration pattern,suggesting substantial fragmentation of the protein constituents (FIGS.2A and B). From the migration patterns of the SPS formulated vaccines itcan be seen that the chitosan-containing preparations liquid SPS T final(before SD), SD SPS T final and SD SPS T final 25 kGy (FIGS. 2A and B,lanes 2 to 4) resulted in a long smear overlaying individual bands. Itis well-known that chitosan can bind to lipids [29]. Thus, chitosan-HClmight bind to the residual fragments of the lipid membrane of the splitvaccine in these formulations, which may cause this effect. There was noloss of any protein band and particularly of haemagglutinin in SPSprotected vaccines after spray-drying and irradiation.

EXAMPLE 4: ANALYSIS OF STORAGE STABILITY SDS PAGE Electrophoresis

After storage times of one month (FIGS. 2C and D; lanes 2 to 5) andthree months (FIGS. 2E and F; lanes 2 to 5) at 2 to 8° C. and at 25° C.(rH 60%), the analysis showed no changes in the migration pattern of theSD SPS T final and SD SPS T final 25 kGy compared to the correspondingmigration pattern at t=0 (FIGS. 2A and B; lanes 2 to 4). In the case ofSD M 25 kGy, storage for 1 and 3 months at 25° C. (rH 60%) led to afurther loss of residual protein components of the split vaccine (FIGS.2C, D, E, and F, lane 7) compared to storage at 2 to 8° C. (FIGS. 2C, D,E, and F; lane 6) and time point t=0 (FIGS. 2A and B; lane 7). Thisshows that the SPS-stabilised spray dried and particularly theirradiated formulation remained stable not only upon refrigeratedstorage, but also at 25° C., 60% rH demonstrating increased thermostability.

Dynamic Light Scattering (DLS)

DLS data displays the size of colloidal components and aggregates. Asvisible in FIG. 4a , original Pandemrix consists of colloidal componentsof a certain size distribution, but no large aggregates were visible.After spray drying, aggregates were visible for the SD M formulation asindicated by the second peak at around 5000 nm, but not for the SD SPS Tfinal formulation (FIGS. 4a and b ). After irradiation a slight increasein aggregation can be seen for the SD SPS T final 25 kGy formulation(FIGS. 4a and d ). On storage for one month and three months,respectively, the spray dried and the subsequently irradiatedformulations with SPS did not show an increase in aggregationirrespective of the storage conditions as shown in FIGS. 4c and d . Thisindicates that no destabilising processes take place over storage timewhich would increase aggregation upon redispersion.

Size Exclusion Chromatography

In size exclusion chromatography the highly hydrophobic nature of theprotein led to elution as protein complexes. SEC chromatograms are shownin FIG. 5. The first peak at about 11 ml in the original Pandemrix couldbe assigned to a high molecular weight complex of the proteinconstituents>500 kDa. The second peak at 16 ml may represent the HAtrimer of 255 kDa; it became larger in the SD M and is heavily increasedin the SD M 25 kGy (FIG. 5a ). The third peak could be the HA monomer,which is totally lost in the corresponding irradiated formulation SD M25 kGy (FIG. 5a ). The SD SPS T final exhibits the same peaks of thetrimer and the monomer, but lost the high molecular weight complex(FIGS. 5a and c ) indicating a stable formulation.

In contrast, SD M 25 kGy showed changes in the trimer peak area,especially upon storage at 25° C./60% rH for 1 and 3 months (FIGS. 5aand b ) indicating that the antigen was not stabilised properly leadingto increased aggregation upon redispersion. SEC chromatographs (FIGS. 5cand d ) showed no change in the SD SPS T final or the SD SPS T final 25kGy formulation after storage for 1 and 3 months at either storagecondition compared to the freshly prepared formulation.

Water Content

As storage stability was performed under tightly sealed conditions, aremarkable change would indicate an improper sealing. No such effect wasobserved (FIG. 6).

EXAMPLE 5: IN VIVO ANALYSIS

Throughout the experimental period all animals maintained weight,haemoglobin level and body temperature within the expected range for thespecies. No adverse reactions were observed at the site of injection andno lymphadenopathy was detected by palpation. 21 days post-vaccination,all animals in groups 4 and 5, and five out of six animals in group 2,had sero-converted as shown by an HAI titre≥40 (FIG. 3a ). In contrast,all six animals in group 3 (SD M 25 kGy) remained sero-negative (HAItitres≤20). These observations were confirmed by MN titres which showedgroup 3 sera to be equivalent to mock-vaccinated control sera (MN titres40-80), with all animals in groups 2, 4 and 5 showing MN titres ofbetween 160 and 5120 (FIG. 3b ).

All animals then received a booster vaccination 28 days after primaryvaccination, and sera were taken for analysis 6 and 20 days post-boost.Following boost, all animals in group 3 showed seroconversion by HAI andMN titres. However, mean titres for group 3 remained significantly lowerthan mean titres of groups 2, 4 and 5 in both assays (FIG. 3). Meantitres of groups 2, 4 and 5 were not significantly different from oneanother. Mock-vaccinated animals were boosted with PBS and did notsero-convert. These data demonstrate that spray-drying and irradiationled to >10-fold reduction in mean HAI titre and >15-fold reduction inmean MN titre in the absence of protection; whereas in the presence ofthe inventive solution, there was no significant reduction in titre dueto irradiation. Furthermore, spray-drying of vaccine in the presence ofthe inventive solution had no detrimental effect on immunogenicitycompared to untreated vaccine.

EXAMPLE 6: STORAGE STABILITY OF THE MODEL ENZYME LACTIC DEHYDROGENASE(LDH) AT 50° C. IN THE LIQUID STATE

Further experiments were carried out to show the influence of individualcompounds on the stability of proteins during storage. In addition, adirect comparison with the results described in WO 2009/014774 wascarried out, where a combination of chitosan with trehalose, PluronicF127 and rHSA was allegedly shown to decrease the antigen titre lossupon incubation at 37° C. However, the presence of rHSA in the samplesrenders it impossible to analyse the effect on the above employed splitvirus preparation, as the rHSA would present in an SDS-PAGE analysis atthe same band size as hemagglutinin. To nonetheless enable a directcomparison, the following experiments were carried out using the modelenzyme lactic dehydrogenase (LDH) as proof of concept. LDH is a commonlyemployed model protein employed in the development of spray driedformulations, freeze drying and the effect of storage on this enzyme canconveniently be analysed in enzyme assay readouts.

Materials and Methods

Lactic dehydrogenase (LDH; Sigma-Aldrich, Munich, Germany) wasrecombinantly expressed in E. coli and was used as the model protein,for the reasons detailed above. A 50 mg/ml stock solution of LDH wasprepared by dissolving the lyophilized powder of the enzyme in 10 mMsodium phosphate buffer pH 7.5. For the preparation of the formulationvariants the LDH stock solution was further diluted to an enzymeconcentration of 1 mg/ml with the respective formulations. The LDHformulations were subsequently incubated at 50° C. for 21 days and theenzyme activity of lactic dehydrogenase was measured at the indicatedtime points (t=0; 2; 4; 7; 14; 16; 18; and 21 days).

LDH enzymatic activity was determined by monitoring the decrease inabsorbance of the reduced cofactor NADH at a wavelength of 340 nm and ata temperature of 22° C. upon the enzymatic reaction of pyruvate tolactate. Before measuring the activity, the formulations were diluted toa concentration of 37.5 μg/ml with 100 mM sodium phosphate buffer pH7.5. A reaction mixture of 790 μl sodium phosphate buffer pH 7.5; 100 μlpyruvate stock solution (20 mM) and 100 μl NADH stock solution (1 mM)was prepared and the enzymatic reaction was started by addition of 10 μlLDH dilution (37.5 μg/ml).

Results

The enzymatic activity of LDH after liquid storage at 50° C. wasmeasured at various time points. FIG. 7 summarises the results obtainedfor LDH storage after formulation with one of 4 different SPS variantsin comparison to a buffer formulation. SPS 17 is a mixture of 4 aminoacids, SPS 18 is a mixture of 4 amino acids with trehalose, SPS 19 is amixture of 4 amino acids in combination with chitosan and SPS 20 is amixture of 4 amino acids in combination with trehalose and chitosan.

As is shown in FIG. 7, when employing only a buffer formulation, therewas a significant loss of activity upon storage at 50° C. In contrast,employing any of the 4 SPS variants protected LDH from such a loss ofactivity. Moreover, the addition of chitosan in both cases (see SPS 19and SPS 20) increased the enzymatic activity of LDH upon liquid storagecompared to the same SPS formulations without chitosan (SPS 17 and SPS18).

FIG. 8, left group of bars, shows that a mixture of trehalose andchitosan alone, without amino acids, resulted in a dramatic lossfunction of the enzyme after liquid storage at 50° C. for 16; 18 and 21days. The addition of amino acids to this mixture of trehalose andchitosan, however, was capable of leading to the retention of theenzymatic function during liquid storage of LDH at 50° C. up to 21 days.For a direct comparison with the results described in WO 2009/014774,the main components of WO 2009/014774, namely Pluronic F127 and rHSA,were combined with either trehalose, or with trehalose and chitosan, orwith amino acids, trehalose and chitosan. As is shown in FIG. 9, rightgroup of bars, the mixture of amino acids, trehalose and chitosan withPluronic F127 and HSA led to an increase of the LDH activity between 20and 40% after storage at 50° C. as compared to a mixture of PluronicF127 and HSA with either trehalose alone (left group of bars) or withtrehalose and chitosan (middle group of bars).

Tables:

TABLE 1 Calculated midpoints of thermal denaturation T_(m) from thethermal denaturation plots corresponding to the model protein 300 μg/mlin buffer and in the analysed excipient mixtures in differentconcentrations (order C1 to C8 with increasing concentration). C 1 C 2 C3 C 4 C 5 C 6 C 7 C 8 T_(m) T_(m) T_(m) T_(m) T_(m) T_(m) T_(m) T_(m)Formulation [° C.] [° C.] [° C.] [° C.] [° C.] [° C.] [° C.] [° C.] PBS70.7 70.7 70.7 70.7 70.7 70.7 70.7 70.7 trehalose 73.7 73.2 73.7 73.571.9 72.7 72.8 72.8 chitosan 73.9 74.0 73.9 74.0 74.2 74.9 75.9 75.4 SPS73.2 73.1 n.d. 73.7 73.5 73.5 73.3 73.8 SPS + 72.3 n.d. 73.0 74.4 75.776.6 n.d. 76.0 trehalose SPS + 73.6 73.9 73.9 74.3 75.6 77.5 n.d. 79.3chitosan SPS + 73.4 73.7 73.2 74.2 76.0 78.4 80.9 81.5 trehalose +chitosan

TABLE 2 Calculated midpoints of thermal denaturation T_(m) from thethermal denaturation plots corresponding to the model protein 600 μg/mlin buffer and in the analysed excipient mixtures in differentconcentrations (order C1 to C8 with increasing concentration). C 1 C 2 C3 C 4 C 5 C 6 C 7 C8 T_(m) T_(m) T_(m) T_(m) T_(m) T_(m) T_(m) T_(m)Formulation [° C.] [° C.] [° C.] [° C.] [° C.] [° C.] [° C.] [° C.] PBS70.2 70.2 70.2 70.2 70.2 70.2 70.2 70.2 trehalose 73.6 73.5 74.2 74.273.5 73.0 72.6 73.1 chitosan 73.6 73.7 73.4 73.1 73.3 74.0 75.2 75.4 SPS73.1 72.9 72.8 73.9 72.8 72.4 72.1 73.0 SPS + n.d. 72.8 73.1 n.d. 74.074.4 74.8 75.8 trehalose SPS + 73.5 73.6 73.3 74.1 75.5 78.1 n.d. 78.9chitosan SPS + 73.3 73.1 73.2 73.8 75.7 77.6 80.4 81.5 trehalose +chitosan

TABLE 3 Overview of formulation variants. Formulations for the in vivostudy are marked with an asterisk. SPS HA further Pandemrix (mg/ (μg/matrix components/ formulations mL) mL) (mg/mL) variations irradiationOriginal — 15 — — — Pandemrix* SD M — 15 Mannitol, — No 160 Yes* 25 kGyYes 40 kGy SD T — 15 Trehalose, — No 160 Yes 25 kGy Yes 40 kGy SD SPS80M 80 15 Mannitol, — No 160 SD SPS80 T 80 15 Trehalose, — No 160 Yes 25kGy Yes 40 kGy SD SPS80 T80 80 15 Trehalose, — No 80 SD SPS40 T80 40 15Trehalose, — No 80 Yes 25 kGy Yes 40 kGy SD SPSv1 T 80 15 Trehalose,Variation of No 160 SPS (no Yes 25 kGy hygroscopic Yes 40 kGy aminoacids) SD SPS T 80 15 Trehalose, 2 mg/ml GA + No* final 160 2 mg/ml Yes*25 kGy Chitosan-HCl

TABLE 4 Spray-drying parameters used. Parameters Values Two fluid nozzle1.5 mm inner diameter Inlet air temperature 120 (° C.) Aspirator airflow 35 (m³/h) = 100% Flow rate 5-6 (ml/min) Spray flow rate 470 L/hOutlet air temperature 50-55 (° C.)

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1. A method for producing stabilised vaccines, the method comprising:(a) mixing antigens with a solution comprising: (i) chitosan; (ii) atleast three different amino acids and/or at least one dipeptide ortripeptide; and (iii) a sugar; and (b) drying the mixture obtained in(a).
 2. The method of claim 1, wherein the at least three amino acidsare selected from the groups of (a) amino acids with nonpolar, aliphaticR groups; (b) amino acids with polar, uncharged R groups; (c) aminoacids with positively charged R groups; (d) amino acids with negativelycharged R groups; and (e) amino acids with aromatic R groups.
 3. Themethod of claim 1, wherein the solution comprises at least one aminoacid selected from each group of (a) an amino acid with nonpolar,aliphatic R groups; (b) an amino acid with polar, uncharged R groups;(c) an amino acid with positively charged R groups; (d) an amino acidwith negatively charged R groups; and (e) an amino acid with aromatic Rgroups.
 4. The method according to claim 1, wherein the solutioncomprises at least the amino acids selected from: (a) alanine,glutamate, lysine, threonine and tryptophane; (b) aspartate, arginine,phenylalanine, serine and valine; (c) proline, serine, asparagine,aspartate, threonine, phenylalanine; (d) tyrosine, isoleucine, leucine,threonine, valine; (e) arginine, glycine, histidin, alanine, glutamate,lysine, tryptophane; and (f) alanine, arginine, glycine, glutamate,lysine.
 5. The method according to claim 1, wherein one or more of theamino acids are selected from natural non-proteinogenic amino acids andsynthetic amino acids.
 6. The method according to claim 1, wherein atleast one of the dipeptide(s) in accordance with claim 1(a)(ii) isselected from carnosin, glycyltyrosine, glycylglycine andglycylglutamine.
 7. The method according to claim 1, wherein the sugaris trehalose.
 8. The method according to claim 1, wherein the solutionfurther comprises at least one saponine.
 9. The method according toclaim 1, wherein the antigens are split virus antigens.
 10. The methodaccording to claim 1, wherein the split virus antigens are influenzavirus antigens.
 11. The method according to claim 10, wherein theinfluenza virus is an influenza A virus.
 12. The method according toclaim 10 or 11, wherein the influenza virus is an influenza A H1N1virus.
 13. The method according to claim 1, wherein the step of dryingthe mixture is achieved by a method selected from spray drying,lyophilisation, spray-freeze drying and air drying.
 14. The methodaccording to claim 1, wherein the dried vaccine obtained in step (b) issubsequently sterilized.
 15. The method according to claim 1, whereinthe vaccine is for intramuscular, subcutaneous, intradermal,transdermal, oral, peroral, nasal, and/or inhalative application.